Streptococcus pneumoniae promotes lung cancer development and progression

Summary Streptococcus pneumoniae (SP) is associated with lung cancer, yet its role in the tumorigenesis remains uncertain. Herein we find that SP attaches to lung cancer cells via binding pneumococcal surface protein C (PspC) to platelet-activating factor receptor (PAFR). Interaction between PspC and PAFR stimulates cell proliferation and activates PI3K/AKT and nuclear factor kB (NF-kB) signaling pathways, which trigger a pro-inflammatory response. Lung cancer cells infected with SP form larger tumors in BALB/C mice compared to untreated cells. Mice treated with tobacco carcinogen and SP develop more lung tumors and had shorter survival period than mice treated with the carcinogen alone. Mutating PspC or PAFR abolishes tumor-promoting effects of SP. Overabundance of SP is associated with the survival. SP may play a driving role in lung tumorigenesis by activating PI3K/AKT and NF-kB pathways via binding PspC to PAFR and provide a microbial target for diagnosis and treatment of the disease.


INTRODUCTION
Lung cancer ranks among the most frequent cancers in the world and is the leading cause of cancer-related deaths in men and women. 1 Over 85% of lung cancers are non-small cell lung cancer (NSCLC), which mainly consists of adenocarcinoma (AC) and squamous cell carcinoma (SCC). The underlying mechanisms for the development and progression of NSCLC have not been completely elucidated. The microbiome, defined as the collection of microbiota and their genes, plays an important role in health and diseases. 2 Microbiota aberrations are attributed to tumorigenesis through different mechanisms, such as damage of the local immune barrier, production of bacterial toxins that alter host genome stability, and release of cancer-promoting microbial metabolites. 3 Furthermore, intratumoral microbes may directly affect the growth and metastatic spread of tumor cells. 2 Infection of Human Papilloma virus, Epstein-Barr virus, Helicobacter pylori, Escherichia coli, and Fusobacterium nucleatum can cause human malignancies, including cervical, nasopharyngeal, and gastrointestinal cancers. 2 As the second microbiome habitat behind the alimentary canal in the human body, the respiratory tract harbors abundant microbiota, containing more than 500 different species of bacteria. [4][5][6][7] Patients with lung cancer have lower microbial diversity and altered abundances of particular bacteria compared with cancer-free individuals. 8 16S rRNA gene sequencing-based studies have identified a set of bacterial genera with either higher or lower abundances in lung tumors vs. normal lung tissues. [9][10][11][12][13][14][15][16] Greathouse et al. showed that Acidovorax was abundant in TP53 mutation-positive lung SCC specimens. 17 Tsay et al. found that airway microbiota affected the progression of NSCLC. 14,16 As early as 1868, William Busch reported spontaneous tumor regressions in patients with Streptococcal infections. 18 Since then, mounting evidence suggests that Streptococcus pneumoniae (SP) is associated with human tumors, particularly lung cancer. 14,19,20 However, it remains uncertain whether SP is a causative pathogen in carcinogenesis or only an opportunistic pathogen or simply commensal bacteria associated with the microenvironment. Furthermore, although numerous bacteria have been suggested to cause human tumors, none has been characterized as a major player in lung tumorgenicity. Here we investigate the role of SP in the tumorigenesis of NSCLC and provide the evidence for oncogenic function of microbiota dysbiosis in the development and progression of lung cancer.
Adhesion and invasion of bacteria to host cells are essential to cause diseases. 21 PAFR is a key adhesion receptor for SP in airway cells and significantly upregulated in NSCLC tissue specimens. [22][23][24] To test the role of PAFR in the attachment and invasion of SP to lung cancer cells, we first analyzed expression of PAFR in NSCLC cell lines (H226, H460, and H1299) and a normal lung epithelial cell line (BEAS-2B). H460 and H1299 cell lines had a higher level of PAFR expression compared with H226 cells and BEAS-2B cells ( Figure 1A). We then explored the capability of SP to attach to and invade the cells. SP had a significantly higher level of adhesion and invasion to H460 and H1299 cancer cells compared with H226 cells and BEAS-2B cells ( Figure 1B). Fluorescence in situ hybridization (FISH) showed that SP was enriched in H460 and H1299 cancer cells compared with H226 cells and BEAS-2B cells ( Figure 1C). Furthermore, SP had a higher level of adhesion and invasion to H460 and H1299 cancer cells compared with Enterococcus faecalis (E. faecalis) and heat-killed SP ( Figure 1B). Therefore, SP could selectively attach to and invade the lung cancer cells that had high PAFR activation.
Pneumococcal surface proteins A and C (PspA and PspC) are among the major factors that interact with respiratory epithelial cells. 6,21,25 Particularly, PspC can specifically bind to PAFR on host cells. 6,21,25 Thus, we tested the role of the surface proteins' binding to PAFR in attachment and invasion of SP. PspC-deficient mutant SP lost adhesion and invasion to H460 and H1299 cancer cells, whereas PspA-deficient mutant SP maintained the activities ( Figure 1B). Furthermore, downregulation of PAFR by small interfering RNA (siRNA) in H460 and H1299 cells significantly inhibited attachment and invasion of SP ( Figure 1D). To further inspect whether adhesion and invasion of SP to lung cancer cells is dependent on the binding, H460 and H1299 cells were treated with WEB2086, a PAFR antagonist, followed by SP infection. WEB2086 could suppress adhesion and invasion of SP to the cells in a dose-dependent manner ( Figure 1E). In addition, enforced expression of PAFR in H226 cells increased attachment and invasion of wild-type SP and PspAdeficient mutant SP, not PspC-deficient mutant SP ( Figure 1F). Taken together, adhesion and invasion of SP to cancer cells require binding of PspC to PAFR.

SP promotes the tumorigenicity of lung cancer by integrating PspC and PAFR
Binding of SP to host cells can dysregulate PAFR recycling pathway, leading to the initiation and development of diseases. 6,21 We first determined if SP infection could promote in vitro tumorgenicity of cancer cells. SP stimulated cell proliferation and migration of PAFR-expressing cells, H460 and H1299 cells (All <0.01) (Figures 2A and 2B). However, E. faecalis or heat-killed SP had no stimulatory effect in all NSCLC cells and BEAS-2B cells (Figures 2A and 2B). We further depleted PAFR in H1299 and H460 using PAFR-siRNA and then incubated the cells with SP ( Figure 2C). The depletion of PAFR in H460 and H1299 cells significantly reduced the effect of SP on cell proliferation ( Figure 2D). The depletion of PAFR in H460 and H1299 cancer cells inhibited the effect of SP on cell migration ( Figure 2E). Furthermore, WEB2086, the PAFR antagonist, repressed the tumor-promoting effects of SP on the cells (Figures 2F and 2G). In addition, enforced activation of PAFR in H266 cancer cells increased the SP-induced tumorigenicity ( Figures 2H and  2I). Therefore, SP infection could promote malignancy in NSCLC cells in a PAFR-dependent manner.
To determine if the integration of PspC and PAFR is essential for the tumor-promoting effects of SP, we treated cancer cells with wild-type, PspC-or PspA-deficient mutant SPs, respectively. The PAFR-expressing cells (H226 and H1299) infected with wild-type strain had higher cell proliferation and migration compared with the cells treated with PspC-deficient mutant SP (Figures 2A and 2B). Furthermore, H460 and H1299 cells infected with PspA-deficient mutant SP reserved the tumor-promoting effects of SP (Figures 2A  and 2B). However, H226 cells infected with all wild-type and mutant SPs did not display elevated cell proliferation and migration (Figures 2A and 2B). Altogether, SP promotes lung tumorigenesis via integrating PspC and PAFR.
SP infection contributes to lung tumorigenesis by stimulating phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and NF-kB signaling pathways PAFR is a direct regulator of PI3K/AKT and nuclear factor kB (NF-kB) signaling pathways whose hyperactivations play essential roles in tumor development and progression. 14,[26][27][28][29][30] We therefore evaluated expression of key molecular signatures of the pathways in cancer cells incubated with or without SP. SP effectively activated PI3K, AKT, ll OPEN ACCESS Figure 1. SP attaches to and invades lung cancer cells via binding PspC to PAFR (A) PAFR expression was determined in cancer cell lines (H226, H460, and H1299) and a normal lung epithelial cell line (BEAS-2B) by Western blot. GAPDH was used as the loading control. Band intensity was determined by using ImageJ, and the ratio of each band was normalized to the corresponding GAPDH and shown below each band. H460 and H1299 cells had a higher level of PAFR expression compared with H226 cells and BEAS-2B cells. Data presented as mean G SEM (n = 3); *p < 0.01 by one-way ANOVA. (B) SP adhered to and invaded the PAFR-expressing cells (H226 and H1299). Bacteria were added to cells at a multiplicity of infection (MOI) of 10 for 1 h. The PspC-deficient mutant SP and heat-killed SP were defective for attachment and invasion compared to wild-type SP and the PspA-deficient mutant SP. E. faecalis did not attach to and invade lung cancer cells. Cells treated with PBS were used as negative controls. Data presented as mean G SEM (n = 3); *p < 0.01 by one-way ANOVA. (C) FISH analysis of SP using an Alexa Fluor 594-conjugated specific probe (Red) to SP. 4 0 ,6-diamidino-2-phenylindole (DAPI) was used to visualize nuclear DNA of cells. Original magnification, X400. Three independent experiments were performed with consistent results. H460 and H1299 cells showed positive staining for SP (Red signals). Scale bar, 10 mm. (D) The depletion of PAFR in H460 and H1299 cells by using siRNA reduced attachment and invasion of SP. Data presented as mean G SEM (n = 3); *p < 0.01 by one-way ANOVA. (E) The PAFR inhibitor, WEB2086, suppressed attachment and invasion of SP to H460 and H1299 cells in a dose-dependent manner (10, 30, and 60 mM and 1,000 mM WEB2086 were used). *p < 0.01. (F) Enforced expression of PAFR in H226 cells increased attachment and invasion of wild-type SP and PspA-deficient mutant SP, but not PspC-deficient mutant SP. All the results are presented as the mean G SD of three different experiments with triplicates. Data presented as mean G SEM (n = 3); *p < 0.01 by one-way ANOVA. iScience Article and NF-kB in H460 and H1299 cells ( Figure 3A). However, the elimination of PAFR in H460 and H1299 cells decreased the SP-induced coactivation of PI3K, AKT, and NF-kB ( Figure 3B). Furthermore, the deletion of PI3K, AKT, or NF-kB reduced the SP-stimulated cell proliferation in the cancer cells ( Figure 3C). It is well established that NF-kB mediates induction of pro-inflammatory cytokines and plays tumor-promoting role of immune and inflammatory responses. 25 Figure 3D). In contrast, the abolition of PAFR in H460 and H1299 cells decreased the SP-induced elevation of the cytokines (Figures S1A and S1B). Furthermore, H226 cells with forced expression of PAFR exhibited an increased level of PI3K, AKT, NF-kB, and the pro-inflammatory cytokines when treated with SP ( Figures 3E and 3F). In addition, SP did not stimulate the pro-inflammatory cytokines in the H460 and H1299 cells with NF-kB depletion ( Figures S2A and S2B). Altogether, SP infection could promote malignancy of lung cancer by activating PI3K/AKT and NF-kB oncogenic pathways and the activation-mediated inflammations.

SP promotes growth of lung tumor and induction of pro-inflammatory cytokines in xenograft animal models
We subcutaneously inoculated NSCLC cells (H460) with SP or PBS in BALB/C nude mice (five mice per group). Sixteen days postinjection, xenograft tumors were observed in all five mice injected with cancer cells treated with SP and in four of the five mice injected with cancer cells treated with PBS ( Figure 4A). Furthermore, the tumors generated from cancer cells treated with SP were significantly larger compared to those created from cancer cells with PBS at the end of observation (days 28) ( Figure 4B) (60.04 G 14.11 mm 3 vs. 33.54 G 9.02 mm 3 , p = 0.0008). In addition, the tumors created from cancer cells treated with SP had higher levels of PAFR, PI3K, AKT, and NF-kB compared with the ones generated from cancer cells treated with PBS ( Figure 4C). Moreover, the xenograft tumors created from the cancer cells treated with SP showed a higher level of pro-inflammatory cytokines (IL-4b, IL-6, IL-8, IL-11, IL-12, TNF-a, and transforming growth factor b [TGF-b]) compared with those generated from cancer cells treated with PBS ( Figure 4D). The xenograft tumors generated from cancer cells infected with SP exhibited a higher level of proliferative marker (KI-67) compared to the controls ( Figure 4C). The findings in ectopic xenograft mouse models are consistent with the above in vitro observations and further support the driving role of SP in lung tumorigenicity.

SP promotes the development of lung cancer and induces pro-inflammatory cytokines in a tobacco carcinogen-induced mouse lung cancer A/J model
Lung cancer is smoking-related disease. 1 Tobacco carcinogen could induce lung cancer in animal models. 31 To assess the role of SP infection in the development of NSCLC, A/J mice exposed to 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanone (NNK), a tobacco carcinogen, were treated with SP alone   Figure 5F). To assess lung tumor burden, we euthanized 6 mice at week 24 with an overdose of CO2, harvested lungs, counted tumors, and measured the sizes. NNK-mice with SP administration had a significant increase in the number of lung tumors compared with mice without SP treatment or mice treated with SP and WEB2086 (All p < 0. 001) (5G). Furthermore, lung tumors in NNK-mice with SP administration were larger than those in mice without SP treatment or NNK-mice treated with SP and WEB2086 ( Figure 5I). Mice treated with SP had a shorter survival period compared to mice without SP treatment or mice treated with SP and WEB2086 (p < 0.05) ( Figure 5J). The observations support that SP infection could initiate the development of lung tumors induced by the tobacco carcinogen and PAFR antagonist might prevent the tumorpromoting effects of SP.

Overabundance of SP correlates with high expression of PAFR in human NSCLC tissues and indicates poor clinical outcomes
To investigate clinical significance of SP infection, we determined DNA abundance of SP and RNA expression of PAFR by using ddPCR in 86 surgical NSCLC tissues and the paired normal lung tissues (Table 1). Both Streptococcus and PAFR displayed a higher level in lung tumor tissues compared with the corresponding noncancerous lung specimens (All p < 0.01) (Figures 6A and 6B). There was significant correlation between SP abundance and PAFR expression level in the lung tumor tissues (r = 0.758, p = 0.001) (Figure 6C). Furthermore, levels of SP in the tissue specimens were associated with advanced NSCLC stage (p = 0.026) but not with patient age, sex, or tumor histological type (all p > 0.05) (Table S1). In addition, univariate and multivariate analyses showed that abundance of SP, expression of PAFR, patient age, and tumor stage of NSCLC were significantly associated with disease-specific survival time of the patients (All p < 0.05) (Tables S2 and S3). Moreover, the patients with NSCLC were classified into two groups according  Figure 6D).

DISCUSSION
NSCLC is the leading cause of cancer-related deaths in men and women. 1 Although numerous bacterial aberrations are observed in lung tumors, [8][9][10][11][12][13][14][15]17 the causative role and molecular mechanism in promoting lung tumorigenesis remain unestablished. Here we find that SP infection could promote tumorigenicity of NSCLC by increasing the cell proliferation and migration. The tumor-promoting effect of SP is confirmed in ectopic xenograft mouse models. Furthermore, tobacco smoke carcinogen-treated A/J mice that are administrated with SP develop more lung tumors and have shorter survival times compared with mice treated with the carcinogen alone. In addition, SP is abundant in human lung tumor tissues in a manner of a stepwise increase from the early to advanced stages. Therefore, we report the evidence for oncogenic function of SP infection, instead of being simply a passenger, in the development and progression of NSCLC.
We investigate the underlying mechanism of SP infection in promoting carcinogenesis of NSCLC. SP selectively attaches to and invades PAFR-expressing lung cancer cells and further stimulates cell proliferation and migration in a PAFR-dependent manner. PAFR is associated with early malignant transformation and tumor metastasis of NSCLC. 32 PspA and PspC are two major pneumococcal surface proteins that play crucial roles in host cell attachment. 25 We find that PspC-deficient mutant SP loses the adhesion and invasion to PAFR-expressing lung cancer cells, suggesting that SP attachment and invasion to cancer cells require binding of PspC to PAFR. Furthermore, either PspC-deficient mutant SP or PAFR knockdown inhibits SP from binding and invading and hence abolishes the subsequent cell proliferation and migration. In addition, WEB2086, a PAFR antagonist, could inhibit SP from its binding and invading to cancer cells and obliterates the tumor-promoting effects. However, cancer cells infected with PspA-deficient mutant SP maintain the effects. Therefore, stimulating function of SP infection to promote lung tumorigenesis requires PsPC and PAFR and their interaction. SP might provide a preventive or therapeutic target for individuals at high risk to develop lung cancer or therapeutics of the disease.
PAFR directly regulates PI3K/AKT and NF-kB signaling pathways. 29,30 The PI3K/AKT pathway is one of the most frequently over-activated intracellular pathways by acting on downstream target proteins and contributes to the carcinogenesis, proliferation, invasion, and metastasis of tumor cells. 29 Furthermore, NF-kB plays an essential role in the cellular environment, immunity, inflammation, death, and cell proliferation. 30 Herein, we find that SP infection upregulates PI3K/AKT and NF-kB in PAFR-expressing lung cancer cells. Furthermore, reduced or forced expression of PAFR efficiently abolishes or increases the tumor-promoting function of SP, respectively. In addition, the deletion of PI3K, AKT, or NF-kB in cancer cells diminishes the effects of SP infection on the cell proliferation and migration. Consistently, SP infection promotes in vivo carcinogenicity of lung tumor xenografts that display high activations of PI3K/AKT and NF-kB oncogenic pathways. The mechanistic investigation is verified with tobacco smoke carcinogen-induced lung cancer animal models, whereby SP infection contributes to the development of lung cancer via the PI3K/AKT and NF-kB oncogenic pathways. Furthermore, PAFR antagonist could reduce SP-mediated PI3K/AKT, NF-kB, and cell proliferation in the lung cancer animal model, further supporting that the PspC and PAFR interaction is essential for the tumor-promoting effect of SP. Therefore, SP infection could play an oncogenic role in lung carcinogenesis by activating PI3K/AKT and NF-kB oncogenic pathways via binding PspC to PAFR.
Bacterial infection causes chronic inflammation, which leads to tumor development and progression. 14,16,27,29,30,33 Particularly, NF-kB activation plays tumor-promoting role of immune and inflammatory responses. 30 Our data show that pro-inflammatory cytokines are elevated in cancer cells infected with SP. Interestingly, the elimination of NF-kB significantly reduces pro-inflammatory cytokines in cancer The high abundance of SP is frequently observed in 86 human lung tumor tissues, regardless of histological subtype. Furthermore, SP abundance is positively associated with levels of PAFR and stages of NSCLC and inversely correlated with disease-specific survival of the patients with lung cancer. The results obtained from the clinical specimens provide additional evidence to support that SP infection plays a crucial role in lung cancer development and progression. Although we cannot exclude that SP infection may directly contribute to the death of lung cancer patients, our finding is consistent with recent report that the microbes promote tumor progression and metastasis, 38 which is major cause for the death of lung cancer patients.
In sum, we identify SP as an oncogenic driver to promote the development and progression of lung cancer through interaction of its surface protein PspC with PAFR. The PspC-PAFR interaction initiates SP adhesion and activates PI3K/AKT and NF-kB signaling pathways and the activation-mediated inflammatory responses and hence contributes to lung tumorigenicity. The discoveries would open new horizons to target

Limitations of the study
First, it is well known that actionable mutations, such as epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK), lead to unregulated proliferation and survival of tumor cells and associated with advanced NSCLC stages. It might be interesting to evaluate the collection between status of the actionable mutations and bacterial abundances in lung cancer. Because most of the actionable mutation statuses are not available from the patients, in this present study, we are not able to perform this analysis. However, we are not collecting new lung tumor specimens with the associated actionable mutations to investigate the correlation between SP abundance and actionable mutations and patients' responses to treatments. Second, clinically, the most common immediate cause of death of lung cancer patients is the extensive widespread metastases. The microbes can travel through the circulatory system with the cancer cells and play critical roles in tumor metastasis. 38 Therefore, SP could modulate and promote cancer cell survival, interact with the immune system, and contribute to tumor metastasis, ultimately causing the death of lung cancer patients. Other causes for the death of lung cancer patients include infection. Therefore, we cannot exclude that SP infection may directly contribute to the death of lung cancer patients. However, how many of lung cancer patients died directly from bacterial infection are not available in this current study. We will perform a different study to investigate if SP infection directly causes the death of lung cancer patients by recruiting new lung cancer cases and following their outcomes.   iScience Article iScience Article (F-5 0 -GGTGACTTGGCAGTGCTTTG and R-5 0 -CACGTTGCACAGGAAGTTGG). 63 Copies/mL of PAFR mRNA was directly determined by using the software of the ddPCR system with Poisson distribution analysis.

Tumorigenicity sssay in nude mice
The animal study was performed with the approval of the University of Maryland under code IACUC# 0516007. Six-week-old male nude mice (BALB/C) were purchased from Charles River Laboratory (Wilmington, MA) and randomly divided into three groups (5 mice per group). Mice in group 1 were injected subcutaneously with 1 3 10 6 NSCLC cells incubated with 50 mL SP or PBS. Mice in group 2 and 3 were injected with 1 3 10 6 NSCLC cells only or PBS. All mice were intraperitoneally injected with 150 mg/kg piperacillin (Pfizer, New York, NY) and raised in the specific pathogenÀfree conditions with autoclaved food and water. The tumor volumes were monitored three times per week using the formula V = (length [mm]) X (width [mm]) 2 3 0.52. 64 The tumor size was represented by mean G standard deviation (SD) mm 3 . Mice were euthanized on day 28 under deep anesthesia with pentobarbital (Sigma-Aldrich). The tumor weight was measured and then used for downstream molecular analysis.

A tobacco carcinogen-induced mouse lung cancer model
Six-week-old female A/J mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and housed in the specific-pathogen-free animal quarters of Animal Core Facility, University of Maryland. After 1 week of accommodation, mice were randomly divided into four groups (12 mice per group). Mice in group 1 were treated with PBS only. Mice in groups 2, 3 and 4 were treated with two doses of 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanone (NNK) (100 mg/kg, i.p.) at an interval of a week apart. After the second NNK dose, group 3 mice received intranasal instillations of SP (2 3 10 6 CFU) weekly for 14 weeks. Group 4 mice received intranasal instillations of SP (2 3 10 6 CFU) weekly and intraperitoneal injection of WEB2086 at 5 mg/kg weekly. 65 To assess lung tumor burden, we euthanized 6 mice with an overdose of CO2, harvested lungs, counted tumors, and measured the sizes at week 24. Serum samples were collected for analysis of inflammatory cytokines. We dissected lung tumors and the surrounding noncancerous lung tissue for downstream molecular analysis. The remaining mice in each group were monitored regularly until spontaneous death occurred.
Micro-CT images of the tobacco carcinogen-induced mouse lung cancer A/J model Micro-CT images were taken as previously described. 66,67 Briefly, mice were anesthetized, endotracheally intubated, connected to a Flexivent ventilator (Scireq, Montreal, Canada) and administrated isoflurane at 2% concentration until complete relaxation. The mice were then scanned with a Micro-CAT II (Siemens Pre-Clinical Solutions, Knoxville, TN) X-ray micro-CT, with a source voltage of 80 kVp and a current of 500 mA. Seven hundred projections were acquired during 650 ms iso-pressure peak inspiration breath holds, with an exposure time of 450 ms per projection. The average scan time was 32 min, and the dosage was 70.1 cGy per scan as computed by the Dose Calculator software (Siemens Medical Solutions). All images were calibrated to Hounsfield Units using a water phantom. The micro-CT images had 46 mm/pixel isotropic dimensions. The reconstructed 3D images were performed and analyzed by using the Amira 4.1.1 software (Mercury Computer Systems, Inc., Chelmsford, MA) as previously described. 66,68

QUANTIFICATION AND STATISTICAL ANALYSIS
We determined differences of data between the groups by using one-way ANOVA. Comparisons of means among multiple groups were assessed by 1-way analysis of variance tests. We analyzed the relationships between SP abundance and level of PAFR by using linear regression. Spearman correlation analysis was used to determine correlation between SP abundance and demographic and clinical characteristics of the patients. We used the median value as cutoff point. The impacts of clinical parameters were estimated by using univariate or multivariate Cox proportional hazards regression model analysis. The Kaplan-Meier survival curves and log rank tests were performed to determine association of SP abundance with the disease-specific survival time. The statistical analyses were performed by using GraphPad Prism 7 software (GraphPad lnc. San Diego, CA) and IBM SPSS Statistics 20.0 software (IBM lnc, Armonk, NY).